AMXT-1501 targets membrane phospholipids against Gram-positive and -negative multidrug-resistant bacteria

ABSTRACT The rapid proliferation of multidrug-resistant (MDR) bacterial pathogens poses a serious threat to healthcare worldwide. Carbapenem-resistant (CR) Enterobacteriaceae, which have near-universal resistance to available antimicrobials, represent a particularly concerning issue. Herein, we report the identification of AMXT-1501, a polyamine transport system inhibitor with antibacterial activity against Gram-positive and -negative MDR bacteria. We observed minimum inhibitory concentration (MIC)50/MIC90 values for AMXT-1501 in the range of 3.13–12.5 μM (2.24–8.93 μg /mL), including for methicillin-resistant Staphylococcus aureus (MRSA), CR Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. AMXT-1501 was more effective against MRSA and CR E. coli than vancomycin and tigecycline, respectively. Subinhibitory concentrations of AMXT-1501 reduced the biofilm formation of S. aureus and Enterococcus faecalis. Mechanistically, AMXT-1501 exposure damaged microbial membranes and increased membrane permeability and membrane potential by binding to cardiolipin (CL) and phosphatidylglycerol (PG). Importantly, AMXT-1501 pressure did not induce resistance readily in the tested pathogens.


Figure S1 .
Figure S1.Safety of AMXT-1501 for mammalian cells.Assessment of AMXT-1501 toxicity for human erythrocytes (a), with associated OD measurements at 570 nm (b).(c) AMXT-1501 toxicity for J774 mouse monocyte macrophages, Huh7 human liver cancer cells, 293T human renal epithelial cells, HUVECs, and A549 human lung cancer cells were determined with CCK-8 assays.Representative fields are shown in panel a.Data in panel b and c are means with SDs.

Figure S2 .
Figure S2.Pharmacokinetic analysis of AMXT-1501 in mice.The mean plasma concentrations of AMXT-1501 after a single i.p. injection of 20 mg/kg (3 mice per time point).All data are presented as mean ± SD.AUClast: area under the plasma concentration-time curve from time 0 to 24 h; Cmax: maximal plasma concentration; Tmax: time to maximal plasma concentration; HL_Lambda_z: terminal half-life of the drug; The dashed line indicated the the limit of detection of LC-MS.

Figure S7 .
Figure S7.Molecular structure and formula of membrane phospholipids.

Figure S11 .
Figure S11.In vitro induction of AMXT-1501 non-sensitive clones of S. aureus.Three S. aureus strains were subcultured serially in TSB containing AMXT-1501.The concentration of AMXT-1501 was increased successively from an initial concentration of 1/4× MIC.Cells were cultured at each concentration for 3-5 passages before being exposed to the next concentration.Isolates from the last passage of each concentration were picked.Clones were selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.The MICs of AMXT-1501 were determined again.

Figure S12 .
Figure S12.In vitro induction of AMXT-1501 non-sensitive clones of E. coli and K. pneumoniae.Two E. coli and one K. pneumoniae strains were subcultured serially in TSB containing AMXT-1501.The concentration of AMXT-1501 was increased successively from an initial concentration of 1/4× MIC.Cells were cultured at each concentration for 3-5 passages before being exposed to the next concentration.Isolates from the last passage of each concentration were picked.Clones were selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.The MICs of AMXT-1501 were determined again.

Figure S13 .
Figure S13.In vitro induction of linezolid non-sensitive clones of S. aureus.Three S. aureus strains were subcultured serially in TSB containing linezolid.The concentration of linezolid was increased successively from an initial concentration of 1/4× MIC.Cells were cultured at each concentration for 3-5 passages before being exposed to the next concentration.Isolates from the last passage of each concentration were picked.Clones were selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.The MICs of linezolid were determined again.

Figure S14 .
Figure S14.In vitro induction of tigecycline non-sensitive clones of E. coli and K. pneumoniae.Two E. coli and one K. pneumoniae strains were subcultured serially in TSB containing tigecycline.The concentration of tigecycline was increased successively from an initial concentration of 1/4× MIC.Cells were cultured at each concentration for 3-5 passages before being exposed to the next concentration.Isolates from the last passage of each concentration were picked.Clones were selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.The MICs of tigecycline were determined again.

Table S2 .
Potency of AMXT-1501 in combination with different classes of antimicrobials against the CR Enterobacteriaceae E. coli ECO2219.

Table S3 .
Mutations in AMXT-1501 non-sensitive clone of S. aureus YUSA145 strain detected by the whole-genome sequencing.